Project Summary Research in my laboratory seeks to address the question of what large macromolecular complexes exist inside cells and how their function is determined by their structure and location. To accomplish this goal, we use electron cryotomography (ECT) to image native cellular structures in 3D inside intact cells, carrying out structural biology in vivo. Projects in the lab fall into three categories: microbial cell biology, structural biology of HIV, and cryo-EM methods development. The HIV work is complementary to our other projects in that it drives and is driven by the same technology development, but it is funded separately. In microbial cell biology, in the next five years we aim to advance our understanding of: (1) the architectures and relationships of bacterial secretion systems; (2) the mechanisms of cell division across all three kingdoms of life; and (3) the bacterial cytoskeleton and DNA-segregating filaments. Driving these projects, in cryo-EM methods development, we aim to: (1) expand the throughput and automation of ECT; (2) apply cryogenic focused ion beam-milling to expand the reach of ECT to any cell, regardless of size; and (3) use our ECT measurements to inform the development of realistic coarse-grained models of biological processes to develop and test mechanistic hypotheses.